1. Field of the Invention
The present invention is related to the fields of molecular biology, developmental biology, physiology, neurobiology, endocrinology and medicine.
2. Related Art
PTH is the principal regulator of blood calcium levels and mediates this action through binding to PTH-1 receptors on bone and kidney cells (Kronenberg, H. M., et al., in “Handbook of Experimental Pharmacology, Springer-Verlag,” Heidelberg (1993)). This receptor also responds to PTH-related peptide, a factor which plays a role in embryonic bone development and is the causative agent of hypercalcemia of malignancy (Lanske, B., et al., Science 273:663–666 (1996)). PTH and PTHrP peptides have been shown to have potent anabolic effects on bone, and it is possible, therefore, that PTH-1 receptor agonists could ultimately be used to treat metabolic bone diseases, such as osteoporosis (Dempster, D. W., et al., Endocr Rev. 14(6):690–709 (1994)).
In the fully bioactive PTH(1–34) peptide, the major determinants of receptor-binding affinity reside within amino acids 15 to 34 (Nussbaum, S. R., et al., J. Biol. Chem. 255:10183–10187 (1980); Gardella, T. J., et al., Endocrinology 132(5):2024–2030 (1993); Caulfield, M. P., et al., Endocrinology 127:83–87 (1990); Abou-Samra, A. B., et al., Endocrinology 125:2215–2217 (1989)), which are moderately conserved among PTHs and PTHrPs from various species (Suva, L. J., et al., Science 237(4817):893–896 (1987)). The determinants of receptor activation lie within the more stringently conserved amino-terminal residues, and deletion of these residues yields competitive PTH-1 receptor antagonists (Horiuchi, N., et al., Science 220:1053–1055 (1983); Nutt, R. F., et al., Endocrinology 127:491–493 (1990)). Amino-terminal PTH or PTHrP fragments shorter in length than PTH(1–27) have not previously been found to be biologically active (Rosenblatt, M., Pathobilogy Annual, Raven Press, New York, 11:53–84 (1981); Azarani, A., et al., J. Biol. Chem. 271(25):14931–14936 (1996); Tregear, G. W., et al., Endocrinology 93:1349–1353 (1973)), yet the functional importance and evolutionary conservation of the amino-terminal residues predicts that they directly interact with the receptor.
The PTH-1 receptor couples strongly to the adenylyl cyclase/protein kinase A signaling pathway and, in some settings, to other pathways including those mediated by phospholipase C/protein kinase C and intracellular calcium (About-Samra, A. B., et al., Endocrinology 129:2547–2554 (1991); Jüppner, H., et al., Science 254:1024–1026 (1991); Guo, J. et al., Endocrinology 136:3884–3891 (1995); Hruska, K. A., et al., J. Clin. Invest. 79:230–239 (1987); Donahue, H. J., et al., J. Biol. Chem. 263:13522–13527 (1988)). The PTH-1 receptor is a member of the family B subgroup of G protein-coupled receptors, which also includes the receptors for calcitonin and secretin (Kolakowski, L. F., “GCRDb: A G-Protein-Coupled Receptor Database,” Receptors and Channels 2:1–7 (1994)). Mutagenesis and crosslinking studies have indicated that multiple domains of these receptors contribute to ligand interaction, including the large amino-terminal extracellular domain, the extracellular loops and the transmembrane helices (Jüppner, H., et al., Endocrinology 134:879–884 (1994); Lee, C., et al., Mol. Endo. 9:1269–1278 (1995); Turner, P., et al., J. Bone Min. Res. 12(1):Abstract 121 (1997); Dautzenberg, F., et al., Proc. Natl. Acad. Sci. 95:4941–4946 (1998); Holtmann, M., et al., J. Biol. Chem. 270:14394–14398 (1995); DeAlmeida, V. and Mayo, K., Mol. Endo. 12:750–765 (1998); Stroop, S., et al., Biochem. 34:1050–1057 (1994); Zhou, A., et al., Proc. Natl. Acad. Sci. USA 94:3644–3649 (1997); Bisello, A., et al., J. Biol. Chem. 273:22498–22505 (1998)). Studies using PTH/calcitonin chimeric receptors and hybrid ligands have suggested a general topology of the interaction in which the amino-terminal extracellular domain of the receptor recognizes the carboxyl-terminal binding domain of the ligand, while the “core” region of the receptor containing the seven transmembrane helices and connecting loops recognizes the amino-terminal signaling portion of the ligand (Bergwitz, C., et al., J. Biol. Chem. 271:26469–26472 (1996)). Similar conclusions were derived from earlier receptor chimera studies (Jüppner, H., et al., Endocrinology 134:879–884 (1994); Stroop, S., et al., Biochem. 34:1050–1057 (1994); Gardella, T. J., et al., Endocrinology 135:1186–1194 (1994)) and from recent crosslinking studies with photoreactive PTH analogs (Bisello, A., et al., J. Biol. Chem. 273:22498–22505 (1998); Mannstadt, M., et al., J. Biol. Chem. 273:16890–16896 (1998)).
In the current study we investigate the signaling component of the interaction between PTH and the PTH-1 receptor using a domain-based approach. This approach employs short amino-terminal PTH fragment analogs and a PTH receptor mutant that lacks most of the amino-terminal extracellular domain. The results of cAMP-signaling assays performed with these smaller ligands and receptors demonstrate that the conserved amino-terminal (Kronenberg, H. M., et al., in “Handbook of Experimental Pharmacology, Springer-Verlag,” Heidelberg (1993); Lanske, B., et al., Science 273:663–666 (1996); Dempster, D. W., et al., Endocr Rev. 14(6):690–709 (1994); Nussbaum, S. R., et al., J. Biol. Chem. 255:10183–10187 (1980); Gardella, T. J., et al., Endocrinology 132(5):2024–2030 (1993); M. P. Caulfield et al., Endocrinology 127:83–87 (1990); A. B. Abou-Samra et al., Endocrinology 125:2215–2217 (1989); Suva, L. J., et al., Science 237(4817):893–896 (1987); Horiuchi, N., et al., Science 220:1053–1055 (1983); Nutt, R. F., et al., Endocrinology 127:491–493 (1990); Rosenblatt, M., Pathobilogy Annual, Raven Press, New York, 11:53–84 (1981); Azarani, A., et al., J. Biol. Chem. 271(25):14931–14936 (1996); Tregear, G. W., et al., Endocrinology 93:1349–1353 (1973); About-Samra, A. B., et al., Endocrinology 129:2547–2554 (1991)) segment of PTH functions as an autonomous signaling domain and that this domain interacts with the core region of the receptor.